Gene Panel Sequencing of Primary and Relapsed Pediatric T-ALL Shows That Relapse-Specific Mutations Are Diverse and Mostly Non-Recurrent

Precursor T-cell acute lymphoblastic leukemia (T-ALL) remains one of the major challenges of pediatric oncology, because relapses are frequently refractory to treatment and fatal. We aimed at identifying relapse specific genetic alterations by analyzing a cohort of 147 primary T-ALL patients and of 70 relapsed T-ALL patients with targeted gene panel sequencing. In addition to the analysis of single nucleotide variants (SNVs) and small insertions and deletions (InDels), we made use of the available coverage data to characterize aberrant copy number alterations (CNA).

DNA from bone marrow of these 217 pediatric T-ALL patients was analyzed by gene panel sequencing after target capture with Agilent HaloPlex. In the target capture design, exons of 324 genes were included that had been found before by whole exome sequencing to carry somatic mutations in a pilot set of relapsed T-ALL or that have been reported to be mutated in T-ALL in the literature. We did not analyze corresponding remission samples and did not discriminate between germline and somatic alterations. Only mutations with an allele frequency (AF) > 10% were considered and absence of the mutation in the 1000 Genomes variant catalogue was required. Copy number analysis based on read-depth data identified deletions (DEL) and amplifications (AMP). Direct comparison of CNAs by multiplex ligation-dependent probe amplification (MPLA) and gene panel sequencing was possible for 13 overlapping regions covering 14 genes in 185 samples. Recognition rate by coverage analysis was 98% (256/260) for biallelic alterations and 81% (92/114) for monoallelic alterations found by MLPA.

On average, gene panel sequencing identified 6.7 mutations in initial diagnosis samples (SNVs: 5.2; InDels: 1.5) and 7.9 mutations in relapse samples (SNVs: 5.9; InDels: 2). In the group of primary leukemia and relapse samples, the average AMP/DEL per patient was 8.2 (AMP: 3.2, DEL: 5.0) and 8.8 (AMP: 4.2, DEL: 4.6), respectively. 31 genes were found to be mutated and 46 deleted/amplified in 10 or more patients (see Table 1 and 2).

Table 1: Most commonly mutated genes (SNVs and InDels)

Gene	Total # of mutations	# pts with mutation in primary T-ALL (n=147)	# pts with mutation in relapsed T-ALL (n=70)
NOTCH1	178	88 (60%)	38 (54%)
PHF6	47	24 (16%)	16 (23%)
FBXW7	42	20 (14%)	17 (24%)
OBSCN	35	20 (14%)	10 (14%)
DNM2	28	17 (12%)	10 (14%)
PTEN	34	20 (14%)	4 (6%)
XIRP2	24	19 (13%)	5 (7%)
CDH23	23	11 (7%)	11 (16%)
WT1	36	11 (7%)	8 (11%)
NT5C2	22	1 (1%)	17 (24%)

Table 2: Most common copy number alterations

Amplifications			Deletions
Gene	primary T-ALL (n=147)	relapsed T-ALL (n=64)	Gene	primary T-ALL (n=147)	relapsed T-ALL (n=64)
MYB	9 (6%)	9 (15%)	CDKN2A	102 (70%)	36 (59%)
MYC	11 (8%)	6 (10%)	CDKN2B	83 (57%)	31 (51%)
NRG1	11 (8%)	3 (5%)	MLLT3	28 (19%)	5 (8%)
UNC5D	11 (8%)	3 (5%)	PHIP	18 (12%)	6 (10%)
NCOA2	11 (8%)	3 (5%)	ELOVL4	18 (12%)	5 (8%)
PTK2B	11 (8%)	3 (5%)	MAP3K7	17 (12%)	5 (8%)
FDFT1	11 (8%)	3 (5%)	CASP8AP2	17 (12%)	5 (8%)
ABL1	8 (5%)	3 (5%)	APC	19 (13%)	3 (5%)
CNOT3	8 (5%)	3 (5%)	LEF1	16 (11%)	5 (8%)
SMG8	3 (2%)	7 (10%)	PAX5	15 (10%)	5 (8%)

Potential novel mechanisms of oncogene activation are amplifications of PTK2B, a gene that has been found to be deregulated by fusion in Philadelphia-like BCP-ALL and that is potentially targetable by tyrosine kinase inhibitors, and of MYC, which has long been known to be a key player in T-ALL leukemogenesis and that is amplified in neuroblastoma and medulloblastoma.

Enriched in relapse, we identified mutations in NT5C2 (p=1.4E-08), TP53 (p=0.0006) and CCDC88A (p=0.01), and amplifications of a region on chr 17q represented by the genes CLTC, ABCA5, C17orf80 and SRSF2. MLLT3 deletions were enriched in primary samples (p=0.04), consistent with the observation that MLLT3 deletions confer a lower risk of relapse in patients treated on BFM protocols.

Conclusion

Gene panel sequencing emerges as a suitable tool for a comprehensive genetic characterization of pediatric T-ALL. Within the group of selected genes contained in the panel, CNA were as frequent as point mutations. Only few genes were found to be specifically altered in relapse, indicating that progression to relapse may involve diverse, non-recurrent genetic alterations.